Our view of the acute effects of ethanol on neuronal function has evolved from nonspecific membrane actions to perturbation of specific sites on specific proteins. Several new techniques and approaches make it possible to ask key questions about the interaction of alcohol with brain proteins and to link changes in protein function with specific behavioral actions of alcohol. During the past grant period we developed alkanethiol reagents as alcohol analogs that could be used to covalently label putative alcohol binding sites in proteins of interest. We propose to use this approach to test the hypotheses that ethanol alters the function of glycine and GABAa receptors by binding within water-filled protein cavities, and that occupation of these cavities alters channel gating. Identification of amino acids critical for alcohol action on target proteins should help us to answer the key question: Which behavioral effects of ethanol are due to actions on which specific proteins? The path from critical amino acids to behavioral studies is illustrated by the recent construction of knock in mice in which mutation of a single amino acid in a single GABAa receptor subunit alters a specific behavioral action of benzodiazepines. These types of mutant mice provide a powerful new approach to link a single protein with a specific behavior. A major goal of the current proposal is to use this approach to define the importance of glycine and GABAa receptors in alcohol actions in vivo. In addition to the knock in approach, the more conventional knock out or null mutant mice will also be used to provide valuable information about the role of specific proteins in drug action and will be employed in this project. The long-range goal of this work is to define key protein sites that can serve as targets for new therapies for alcohol reinforcement, dependence and relapse.